Mitochondrial dosimeter

This example demonstrates detection of mt mutations in tissue samples.

To determine whether mt mutations could be identified in cancer other than colorectal cancer, we studied primary bladder (n=14), head and neck (n=13), and lung (n=14) tumors (5). Eighty percent of the mt genome of all the primary tumor samples was PCR-amplified (6) and sequenced manually (FIG. 1 ). Tumor mtDNA was compared to mtDNA from paired blood samples in all cases, and mtDNA from corresponding normal tissue when available (7). Of the 292 sequence variants detected, 196 were previously recorded polymorphisms (2, 8), while 57 were novel polymorphisms (Table 3). The remaining 39 variants were acquired (somatic) mutations identified in 64% (9/14) of the bladder cancer, 46% (6/13) of the head and neck cancer, and 43% (6/14) of the lung cancer patients (Table 2). Most of these mutations were T-to-C and G-to-A base transitions, indicating possible exposure to ROS-derived mutagens (9). Similar to the previous observation by Polyak et al. (Table 1; 4), the majority of the somatic mutations identified here were also homoplasmic in nature. In addition, several of the bladder and head and neck cancers studied here (Table 2) had multiple mutations implying possible accumulation of mtDNA damage.

In the bladder tumors, mutation hot spots were primarily in the NADH dehydrogenase subunit 4 (ND4) gene (35%), and in the displacement-loop (D-loop) region (30%). The D-loop region is a critical site for both replication and expression of the mt genome since it contains the leading-strand origin of replication and the major promoters for transcription (10). Many (73%) of the mutations identified within protein-coding regions were silent, except for a (Val→Ala) substitution in the NADH dehydrogenase subunit 3 (ND3) and a 7-amino-acid deletion in cytochrome b (Cyt b). The D-loop region was also commonly mutated in head and neck cancer (67%). Two of the head and neck tumors (22%) contained mutations in the ND4 gene at nucleotide pairs (nps) 10822 and 11150, resulting in amino acid substitutions of Thr→Met and Ala→Thr, respectively. A similar tendency was observed in lung cancers, demonstrating a high concentration of mutations in the D-loop region (70%).

This example demonstrates detection of mt mutations in bodily fluids.

We hypothesized that the homoplasmic nature of these mutations would make them readily detectable in paired bodily fluids. To test this, we extracted and directly amplified mtDNA from urine samples from patients diagnosed with bladder cancer. All three corresponding urine samples available in this study contained the mutant mtDNA derived from tumor tissues. For example, the mtDNA from a urine sample of bladder cancer patient #799 showed the same nucleotide transition (G→A) as seen in the tumor (FIG. 2 A). In all cases, the urine sample contained a relatively pure population of tumor-derived mtDNA, comparable to that of the micro-dissected tumor sample. Consistent with this observation, saliva samples obtained from head and neck cancer patients contained no detectable wild-type (WT) signals (FIGS. 2B, and 2 C). By sequence analysis alone, we were able to detect mtDNA mutations in 67% (6/9) of saliva samples from head and neck cancer patients. In lung cancer cases, we were initially unable to identify mutant bands from paired bronchoalveolar lavage (BAL) fluids because of the significant dilution of neoplastic cells in BAL fluid (11), (FIG. 2 D). We, thus, applied a more sensitive oligonucleotide-mismatch ligation assay to detect mutated mtDNA. As shown in FIGS. 3A and 3B, both lung cancer mutations (arrows) were confirmed in tumor mtDNA with more dilute signals in the corresponding BAL samples, and no signal in the corresponding normal tissues. Again, we detected the majority of mtDNA mutations (8/10) in BAL fluids with the exception of two cases where the ligation assays were not feasible due to the sequence compositions (16183 and 302 nps) adjacent to the mutations.

This example demonstrates the enrichment of mitochondrial mutant DNA in samples relative to nuclear mutant DNA.

To quantitate this neoplastic DNA enrichment, we compared the abundance of mt gene mutations to that of nuclear-encoded p53 mutations in bodily fluids using a quantitative plaque assay. Nuclear and mt fragments that contained a mutated sequence were PCR-amplified and cloned for plaque hybridization (12). Two BAL samples from lung cancer patients were chosen for analysis because they had mutations in both the mt and nuclear genomes. For p53 mutations, the percentages of neoplastic cells among normal cells for patients #1113 and #1140 were 0.1 and 3.0 %, respectively. Remarkably, the abundance of the corresponding mutated mtDNA (MT) was 22% and 52% when compared to the wild-type (WT) mt sequence (FIG. 4 ). This enrichment of mtDNA is presumably due to the homoplasmic nature of these mutations and the high copy number of mt genomes in cancer cells. Enrichment was further suggested by our observations in head and neck paraffin samples where we were able to PCR-amplify 2-3 kb fragments of mtDNA, whereas we were unable to amplify nuclear p53 gene fragments of over 300 bp.

A role for mitochondria in tumorigenesis was implicated when tumor cells were found to have an impaired respiratory system and high glycolytic activity (13,14). Recent findings elucidating the role of mitochondria in apoptosis (15) and the high incidence of mtDNA mutations in colon cancer (4) further support the original hypothesis of mitochondrial participation in the initiation and progression of cancer. Although further investigation is needed to define the functional significance of mt mutations, our data clearly show that those mutations are frequent and present at high levels in all of the tumor types examined.

The homoplasmic nature of the mutated mitochondria remains puzzling. It is estimated that each cell contains several hundred-to-thousands of mitochondria and that each mitochondrion contains 1-10 genomes (16). Conceivably, certain mutated mtDNAs may gain a significant replicative advantage. For example, mutations in the D-loop regulatory region might alter the rate of DNA replication by modifying the binding affinity of important trans-acting factors. Mitochondria that undergo the most rapid replication are likely to acquire more DNA damage, leading to an accumulation of mutational events. Although the mechanism may vary for other mutations (such as silent mutations in the ND4 gene), the accumulation of a particular mtDNA mutation may become more apparent during neoplastic transformation. Even subtle mtDNA mutations may also gain significant replicative advantage, perhaps through interactions with important nuclear factors. Homoplasmic transformation of mtDNA was observed in small populations of cells in other non-neoplastic, but diseased tissues (17), sometimes associated with aging (18). We hypothesize that, in contrast to classic clonal expansion, the process may occur as “pseudoclonal” selection where stochastic segregation of mitochondria (16) together with neoplastic clonal expansion driven by nuclear mutations lead to a homogeneous population of a previously “altered” mitochondrion (FIG. 5 ).

The large number of mt polymorphisms identified here and elsewhere (2) likely reflects the high mutation rate of mtDNA, which is thought to be caused mainly by high levels of ROS (19). In agreement with this, our data imply that constitutive hypervariable areas such as the D-loop region represent somatic mutational hot spots. As further mutations are tabulated in primary tumors, DNA-chip technology can be harnessed to develop high-throughput analyses with sufficient sensitivity (20, 21). Due to its high copy number, mtDNA may provide a distinct advantage over other nuclear genome based methods for cancer and environmental pollutant detection.

Literature Cited

1. R. N. Lightowlers, P. F. Chinnery, D. M. Turnbull, N. Howell, Trends Genet 13, 450 (1997).

2. MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, Ga., USA. URL address: http file type, www host server, gen.emory.edu domain name, mitomap.html directory.

3. D. L. Croteau and V. A. Bohr, J. Biol. Chem. 272, 25409 (1997).

4. K. Polyak et al., Nature Genet . 20, 291 (1998).

5. Paired normal and tumor specimens along with blood and bodily fluids were collected following surgical resections with prior consent from patients in The Johns Hopkins University Hospital. Tumor specimens were frozen and micro-dissected on a cryostat so that the tumor samples contained greater than 70% neoplastic cells. DNA from tumor sections was digested with 1% SDS/Proteinase K, extracted by phenol-chloroform, and ethanol precipitated. Control DNA from peripheral lymphocytes, matched normal tissues, from urine, saliva, and BAL fluid were processed in the same manner as described in (11).

6. Mitochondrial DNAs were amplified using overlapping primers (4) in PCR buffer containing 6% DMSO. Approximately 5-20 ng of genomic DNA was subjected to the step-down PCR protocol: 94° C. 30 sec, 64° C. 1 min, 70° C. 3 min, 3 cycles, 94° C. 30 sec, 61° C. 1 min, 70° C. 3 min, 3 cycles 94° C. 30 sec, 58° C. 1 min, 70° C. 3.5 min 15 cycles, 94° C. 30 sec, 57° C. 1 min, 70° C. 3.5 min, 15 cycles, and a final extension at 70° C. for 5 min. PCR products were gel-purified using a Qiagen gel extraction kit (Qiagen) and sequence reactions were performed with Thermosequenase (Perkin-Elmer) using the cycle conditions (95° C. 30 sec, 52° C. 1 min, and 70° C. 1 min for 25 cycles).

7. Corresponding normal tissues from 4 patients (#874, #915, #1684, and #1678) were available and DNA was extracted from paraffin samples as described previously (9).

8. R. M. Andrew et al., Nature Genet. 23 147, (1999)

9. J. Cadet, M. Berger, T. Douki, J. L. Ravanat, Rev. Physiol. Biochem. Pharmacol. 131, 1 (1997).

8. J. W. Taanman, Biochimica. et. Biophysica. Acta. 1410, 103 (1999).

9. S. A. Ahrendt et al., J. Natl. Cancer Inst. 91, 332 (1999).

10. Subcloning of PCR fragments into phage vector was performed according to the manufacturer's instructions (Stratagene). Titered plaques were plated and subjected to hybridization using tetramethylammonium chloride (TMAC) as a solvent. Positive signals were confirmed by secondary screenings. Oligonucleotides (Oligos) used for this assay were as follows; for patient #1113, p53 and mtDNA sequence alterations were detected using oligos containing either WT-(p53: 5′-GTATTTGGATGTCAGAAACACTT-3′ (SEQ ID NO: 2)/mtDNA: 5′-ACTTCAGGGTCATAAAGCC-3′(SEQ ID NO: 3)) or MT (p53: 5′-GTATTTGGATGTCAGAAACACTT-3′(SEQ ID NO: 4)/mtDNA:5′-ACTTCAGGGCCATAAAGCC-3′(SEQ ID NO: 5)) sequences, respectively. For patient #1140, oligos 5′-ACCCGCGTCCGCGCCATGGCC-3′(SEQ ID NO: 6) and 5′-ACCCGCGTCCTCGCCATGGCC-3′(SEQ ID NO: 7) were used to detect WT and MT sequences, respectively.

11. Warburg, Science 123, 309 (1956).

12. J. W. Shay and H. Werbin, Mut. Res. 186, 149 (1987).

13. D. R. Green and J. C. Reed, Science 281, 1309 (1999).

14. D. C. Wallace, Annu. Rev. Biochem. 61, 1175 (1992).

15. D. C. Wallace, Proc. Natl. Acad. Sci. USA 91, 8746 (1994).

16. K. Khrapko et al., N.A.R. 27, 2434 (1999).

17. C. Richter, J. W. Park, B. N. Ames, Proc. Natl. Acad. Sci. U S A 17, 6465 (1988).

18. M. Chee et al, Science 274, 610 (1996).

19. S. A. Ahrendt et al., Proc. Natl. Acad. Sci. USA 96, 7382 (1999).

20. Fragments containing mutations were PCR-amplified and then ethanol precipitated. For each mutation, discriminating oligonucleotides that contained the mutated base at the 3′ end were designed (TAACCATA-3′ (SEQ ID NO: 8) for patient #915 and TCTCTTACC-3′ (SEQ ID NO: 9) for patient #898). Immediately adjacent [ 32 P] end-labeled 3′ sequences (5′-CACACTACTA-3′ (SEQ ID NO: 10) for patient #915 and 5′-TTTAACCAG-3′ (SEQ ID NO: 11) for patient #898) were used as substrate together with discriminating oligonucleotides for the ligation reaction. After a denaturing step of 95° C. for 5′, the reactions were incubated for 1 hr at 37° in the presence of T4 DNA ligase (Life Technologies), in a buffer containing 50 mM Tris-Cl, 10 mM MgCl 2 , 150 mM NaCl, 1 mM Spermidine, 1 mM ATP, 5 mM DTT, and analyzed on denatured 12% polyacrylamide gels. [Jen et al., Cancer Res. 54, 5523 (1994)].

1

gatcacaggt ctatcaccct attaaccact cacgggagct ctccatgcat ttggtatttt 60

cgtctggggg gtatgcacgc gatagcattg cgagacgctg gagccggagc accctatgtc 120

gcagtatctg tctttgattc ctgcctcatc ctattattta tcgcacctac gttcaatatt 180

acaggcgaac atacttacta aagtgtgtta attaattaat gcttgtagga cataataata 240

acaattgaat gtctgcacag ccactttcca cacagacatc ataacaaaaa atttccacca 300

aaccccccct cccccgcttc tggccacagc acttaaacac atctctgcca aaccccaaaa 360

acaaagaacc ctaacaccag cctaaccaga tttcaaattt tatcttttgg cggtatgcac 420

ttttaacagt caccccccaa ctaacacatt attttcccct cccactccca tactactaat 480

ctcatcaata caacccccgc ccatcctacc cagcacacac acaccgctgc taaccccata 540

ccccgaacca accaaacccc aaagacaccc cccacagttt atgtagctta cctcctcaaa 600

gcaatacact gaaaatgttt agacgggctc acatcacccc ataaacaaat aggtttggtc 660

ctagcctttc tattagctct tagtaagatt acacatgcaa gcatccccgt tccagtgagt 720

tcaccctcta aatcaccacg atcaaaagga acaagcatca agcacgcagc aatgcagctc 780

aaaacgctta gcctagccac acccccacgg gaaacagcag tgattaacct ttagcaataa 840

acgaaagttt aactaagcta tactaacccc agggttggtc aatttcgtgc cagccaccgc 900

ggtcacacga ttaacccaag tcaatagaag ccggcgtaaa gagtgtttta gatcaccccc 960

tccccaataa agctaaaact cacctgagtt gtaaaaaact ccagttgaca caaaatagac 1020

tacgaaagtg gctttaacat atctgaacac acaatagcta agacccaaac tgggattaga 1080

taccccacta tgcttagccc taaacctcaa cagttaaatc aacaaaactg ctcgccagaa 1140

cactacgagc cacagcttaa aactcaaagg acctggcggt gcttcatatc cctctagagg 1200

agcctgttct gtaatcgata aaccccgatc aacctcacca cctcttgctc agcctatata 1260

ccgccatctt cagcaaaccc tgatgaaggc tacaaagtaa gcgcaagtac ccacgtaaag 1320

acgttaggtc aaggtgtagc ccatgaggtg gcaagaaatg ggctacattt tctaccccag 1380

aaaactacga tagcccttat gaaacttaag ggtcgaaggt ggatttagca gtaaactaag 1440

agtagagtgc ttagttgaac agggccctga agcgcgtaca caccgcccgt caccctcctc 1500

aagtatactt caaaggacat ttaactaaaa cccctacgca tttatataga ggagacaagt 1560

cgtaacatgg taagtgtact ggaaagtgca cttggacgaa ccagagtgta gcttaacaca 1620

aagcacccaa cttacactta ggagatttca acttaacttg accgctctga gctaaaccta 1680

gccccaaacc cactccacct tactaccaga caaccttagc caaaccattt acccaaataa 1740

agtataggcg atagaaattg aaacctggcg caatagatat agtaccgcaa gggaaagatg 1800

aaaaattata accaagcata atatagcaag gactaacccc tataccttct gcataatgaa 1860

ttaactagaa ataactttgc aaggagagcc aaagctaaga cccccgaaac cagacgagct 1920

acctaagaac agctaaaaga gcacacccgt ctatgtagca aaatagtggg aagatttata 1980

ggtagaggcg acaaacctac cgagcctggt gatagctggt tgtccaagat agaatcttag 2040

ttcaacttta aatttgccca cagaaccctc taaatcccct tgtaaattta actgttagtc 2100

caaagaggaa cagctctttg gacactagga aaaaaccttg tagagagagt aaaaaattta 2160

acacccatag taggcctaaa agcagccacc aattaagaaa gcgttcaagc tcaacaccca 2220

ctacctaaaa aatcccaaac atataactga actcctcaca cccaattgga ccaatctatc 2280

accctataga agaactaatg ttagtataag taacatgaaa acattctcct ccgcataagc 2340

ctgcgtcaga ttaaaacact gaactgacaa ttaacagccc aatatctaca atcaaccaac 2400

aagtcattat taccctcact gtcaacccaa cacaggcatg ctcataagga aaggttaaaa 2460

aaagtaaaag gaactcggca aatcttaccc cgcctgttta ccaaaaacat cacctctagc 2520

atcaccagta ttagaggcac cgcctgccca gtgacacatg tttaacggcc gcggtaccct 2580

aaccgtgcaa aggtagcata atcacttgtt ccttaaatag ggacctgtat gaatggctcc 2640

acgagggttc agctgtctct tacttttaac cagtgaaatt gacctgcccg tgaagaggcg 2700

ggcataacac agcaagacga gaagacccta tggagcttta atttattaat gcaaacagta 2760

cctaacaaac ccacaggtcc taaactacca aacctgcatt aaaaatttcg gttggggcga 2820

cctcggagca gaacccaacc tccgagcagt acatgctaag acttcaccag tcaaagcgaa 2880

ctactatact caattgatcc aataacttga ccaacggaac aagttaccct agggataaca 2940

gcgcaatcct attctagagt ccatatcaac aatagggttt acgacctcga tgttggatca 3000

ggacatcccg atggtgcagc cgctattaaa ggttcgtttg ttcaacgatt aaagtcctac 3060

gtgatctgag ttcagaccgg agtaatccag gtcggtttct atctacttca aattcctccc 3120

tgtacgaaag gacaagagaa ataaggccta cttcacaaag cgccttcccc cgtaaatgat 3180

atcatctcaa cttagtatta tacccacacc cacccaagaa cagggtttgt taagatggca 3240

gagcccggta atcgcataaa acttaaaact ttacagtcag aggttcaatt cctcttctta 3300

acaacatacc catggccaac ctcctactcc tcattgtacc cattctaatc gcaatggcat 3360

tcctaatgct taccgaacga aaaattctag gctatataca actacgcaaa ggccccaacg 3420

ttgtaggccc ctacgggcta ctacaaccct tcgctgacgc cataaaactc ttcaccaaag 3480

agcccctaaa acccgccaca tctaccatca ccctctacat caccgccccg accttagctc 3540

tcaccatcgc tcttctacta tgaacccccc tccccatacc caaccccctg gtcaacctca 3600

acctaggcct cctatttatt ctagccacct ctagcctagc cgtttactca atcctctgat 3660

cagggtgagc atcaaactca aactacgccc tgatcggcgc actgcgagca gtagcccaaa 3720

caatctcata tgaagtcacc ctagccatca ttctactatc aacattacta ataagtggct 3780

cctttaacct ctccaccctt atcacaacac aagaacacct ctgattactc ctgccatcat 3840

gacccttggc cataatatga tttatctcca cactagcaga gaccaaccga acccccttcg 3900

accttgccga aggggagtcc gaactagtct caggcttcaa catcgaatac gccgcaggcc 3960

ccttcgccct attcttcata gccgaataca caaacattat tataataaac accctcacca 4020

ctacaatctt cctaggaaca acatatgacg cactctcccc tgaactctac acaacatatt 4080

ttgtcaccaa gaccctactt ctaacctccc tgttcttatg aattcgaaca gcataccccc 4140

gattccgcta cgaccaactc atacacctcc tatgaaaaaa cttcctacca ctcaccctag 4200

cattacttat atgatatgtc tccataccca ttacaatctc cagcattccc cctcaaacct 4260

aagaaatatg tctgataaaa gagttacttt gatagagtaa ataataggag cttaaacccc 4320

cttatttcta ggactatgag aatcgaaccc atccctgaga atccaaaatt ctccgtgcca 4380

cctatcacac cccatcctaa agtaaggtca gctaaataag ctatcgggcc cataccccga 4440

aaatgttggt tatacccttc ccgtactaat taatcccctg gcccaacccg tcatctactc 4500

taccatcttt gcaggcacac tcatcacagc gctaagctcg cactgatttt ttacctgagt 4560

aggcctagaa ataaacatgc tagcttttat tccagttcta accaaaaaaa taaaccctcg 4620

ttccacagaa gctgccatca agtatttcct cacgcaagca accgcatcca taatccttct 4680

aatagctatc ctcttcaaca atatactctc cggacaatga accataacca atactaccaa 4740

tcaatactca tcattaataa tcataatagc tatagcaata aaactaggaa tagccccctt 4800

tcacttctga gtcccagagg ttacccaagg cacccctctg acatccggcc tgcttcttct 4860

cacatgacaa aaactagccc ccatctcaat catataccaa atctctccct cactaaacgt 4920

aagccttctc ctcactctct caatcttatc catcatagca ggcagttgag gtggattaaa 4980

ccaaacccag ctacgcaaaa tcttagcata ctcctcaatt acccacatag gatgaataat 5040

agcagttcta ccgtacaacc ctaacataac cattcttaat ttaactattt atattatcct 5100

aactactacc gcattcctac tactcaactt aaactccagc accacgaccc tactactatc 5160

tcgcacctga aacaagctaa catgactaac acccttaatt ccatccaccc tcctctccct 5220

aggaggcctg cccccgctaa ccggcttttt gcccaaatgg gccattatcg aagaattcac 5280

aaaaaacaat agcctcatca tccccaccat catagccacc atcaccctcc ttaacctcta 5340

cttctaccta cgcctaatct actccacctc aatcacacta ctccccatat ctaacaacgt 5400

aaaaataaaa tgacagtttg aacatacaaa acccacccca ttcctcccca cactcatcgc 5460

ccttaccacg ctactcctac ctatctcccc ttttatacta ataatcttat agaaatttag 5520

gttaaataca gaccaagagc cttcaaagcc ctcagtaagt tgcaatactt aatttctgta 5580

acagctaagg actgcaaaac cccactctgc atcaactgaa cgcaaatcag ccactttaat 5640

taagctaagc ccttactaga ccaatgggac ttaaacccac aaacacttag ttaacagcta 5700

agcaccctaa tcaactggct tcaatctact tctcccgccg ccgggaaaaa aggcgggaga 5760

agccccggca ggtttgaagc tgcttcttcg aatttgcaat tcaatatgaa aatcacctcg 5820

gagctggtaa aaagaggcct aacccctgtc tttagattta cagtccaatg cttcactcag 5880

ccattttacc tcacccccac tgatgttcgc cgaccgttga ctattctcta caaaccacaa 5940

agacattgga acactatacc tattattcgg cgcatgagct ggagtcctag gcacagctct 6000

aagcctcctt attcgagccg agctgggcca gccaggcaac cttctaggta acgaccacat 6060

ctacaacgtt atcgtcacag cccatgcatt tgtaataatc ttcttcatag taatacccat 6120

cataatcgga ggctttggca actgactagt tcccctaata atcggtgccc ccgatatggc 6180

gtttccccgc ataaacaaca taagcttctg actcttacct ccctctctcc tactcctgct 6240

cgcatctgct atagtggagg ccggagcagg aacaggttga acagtctacc ctcccttagc 6300

agggaactac tcccaccctg gagcctccgt agacctaacc atcttctcct tacacctagc 6360

aggtgtctcc tctatcttag gggccatcaa tttcatcaca acaattatca atataaaacc 6420

ccctgccata acccaatacc aaacgcccct cttcgtctga tccgtcctaa tcacagcagt 6480

cctacttctc ctatctctcc cagtcctagc tgctggcatc actatactac taacagaccg 6540

caacctcaac accaccttct tcgaccccgc cggaggagga gaccccattc tataccaaca 6600

cctattctga tttttcggtc accctgaagt ttatattctt atcctaccag gcttcggaat 6660

aatctcccat attgtaactt actactccgg aaaaaaagaa ccatttggat acataggtat 6720

ggtctgagct atgatatcaa ttggcttcct agggtttatc gtgtgagcac accatatatt 6780

tacagtagga atagacgtag acacacgagc atatttcacc tccgctacca taatcatcgc 6840

tatccccacc ggcgtcaaag tatttagctg actcgccaca ctccacggaa gcaatatgaa 6900

atgatctgct gcagtgctct gagccctagg attcatcttt cttttcaccg taggtggcct 6960

gactggcatt gtattagcaa actcatcact agacatcgta ctacacgaca cgtactacgt 7020

tgtagcccac ttccactatg tcctatcaat aggagctgta tttgccatca taggaggctt 7080

cattcactga tttcccctat tctcaggcta caccctagac caaacctacg ccaaaatcca 7140

tttcactatc atattcatcg gcgtaaatct aactttcttc ccacaacact ttctcggcct 7200

atccggaatg ccccgacgtt actcggacta ccccgatgca tacaccacat gaaacatcct 7260

atcatctgta ggctcattca tttctctaac agcagtaata ttaataattt tcatgatttg 7320

agaagccttc gcttcgaagc gaaaagtcct aatagtagaa gaaccctcca taaacctgga 7380

gtgactatat ggatgccccc caccctacca cacattcgaa gaacccgtat acataaaatc 7440

tagacaaaaa aggaaggaat cgaacccccc aaagctggtt tcaagccaac cccatggcct 7500

ccatgacttt ttcaaaaagg tattagaaaa accatttcat aactttgtca aagttaaatt 7560

ataggctaaa tcctatatat cttaatggca catgcagcgc aagtaggtct acaagacgct 7620

acttccccta tcatagaaga gcttatcacc tttcatgatc acgccctcat aatcattttc 7680

cttatctgct tcctagtcct gtatgccctt ttcctaacac tcacaacaaa actaactaat 7740

actaacatct cagacgctca ggaaatagaa accgtctgaa ctatcctgcc cgccatcatc 7800

ctagtcctca tcgccctccc atccctacgc atcctttaca taacagacga ggtcaacgat 7860

ccctccctta ccatcaaatc aattggccac caatggtact gaacctacga gtacaccgac 7920

tacggcggac taatcttcaa ctcctacata cttcccccat tattcctaga accaggcgac 7980

ctgcgactcc ttgacgttga caatcgagta gtactcccga ttgaagcccc cattcgtata 8040

ataattacat cacaagacgt cttgcactca tgagctgtcc ccacattagg cttaaaaaca 8100

gatgcaattc ccggacgtct aaaccaaacc actttcaccg ctacacgacc gggggtatac 8160

tacggtcaat gctctgaaat ctgtggagca aaccacagtt tcatgcccat cgtcctagaa 8220

ttaattcccc taaaaatctt tgaaataggg cccgtattta ccctatagca ccccctctac 8280

cccctctaga gcccactgta aagctaactt agcattaacc ttttaagtta aagattaaga 8340

gaaccaacac ctctttacag tgaaatgccc caactaaata ctaccgtatg gcccaccata 8400

attaccccca tactccttac actattcctc atcacccaac taaaaatatt aaacacaaac 8460

taccacctac ctccctcacc aaagcccata aaaataaaaa attataacaa accctgagaa 8520

ccaaaatgaa cgaaaatctg ttcgcttcat tcattgcccc cacaatccta ggcctacccg 8580

ccgcagtact gatcattcta tttccccctc tattgatccc cacctccaaa tatctcatca 8640

acaaccgact aatcaccacc caacaatgac taatcaaact aacctcaaaa caaatgataa 8700

ccatacacaa cactaaagga cgaacctgat ctcttatact agtatcctta atcattttta 8760

ttgccacaac taacctcctc ggactcctgc ctcactcatt tacaccaacc acccaactat 8820

ctataaacct agccatggcc atccccttat gagcgggcac agtgattata ggctttcgct 8880

ctaagattaa aaatgcccta gcccacttct taccacaagg cacacctaca ccccttatcc 8940

ccatactagt tattatcgaa accatcagcc tactcattca accaatagcc ctggccgtac 9000

gcctaaccgc taacattact gcaggccacc tactcatgca cctaattgga agcgccaccc 9060

tagcaatatc aaccattaac cttccctcta cacttatcat cttcacaatt ctaattctac 9120

tgactatcct agaaatcgct gtcgccttaa tccaagccta cgttttcaca cttctagtaa 9180

gcctctacct gcacgacaac acataatgac ccaccaatca catgcctatc atatagtaaa 9240

acccagccca tgacccctaa caggggccct ctcagccctc ctaatgacct ccggcctagc 9300

catgtgattt cacttccact ccataacgct cctcatacta ggcctactaa ccaacacact 9360

aaccatatac caatgatggc gcgatgtaac acgagaaagc acataccaag gccaccacac 9420

accacctgtc caaaaaggcc ttcgatacgg gataatccta tttattacct cagaagtttt 9480

tttcttcgca ggatttttct gagcctttta ccactccagc ctagccccta ccccccaatt 9540

aggagggcac tggcccccaa caggcatcac cccgctaaat cccctagaag tcccactcct 9600

aaacacatcc gtattactcg catcaggagt atcaatcacc tgagctcacc atagtctaat 9660

agaaaacaac cgaaaccaaa taattcaagc actgcttatt acaattttac tgggtctcta 9720

ttttaccctc ctacaagcct cagagtactt cgagtctccc ttcaccattt ccgacggcat 9780

ctacggctca acattttttg tagccacagg cttccacgga cttcacgtca ttattggctc 9840

aactttcctc actatctgct tcatccgcca actaatattt cactttacat ccaaacatca 9900

ctttggcttc gaagccgccg cctgatactg gcattttgta gatgtggttt gactatttct 9960

gtatgtctcc atctattgat gagggtctta ctcttttagt ataaatagta ccgttaactt 10020

ccaattaact agttttgaca acattcaaaa aagagtaata aacttcgcct taattttaat 10080

aatcaacacc ctcctagcct tactactaat aattattaca ttttgactac cacaactcaa 10140

cggctacata gaaaaatcca ccccttacga gtgcggcttc gaccctatat cccccgcccg 10200

cgtccctttc tccataaaat tcttcttagt agctattacc ttcttattat ttgatctaga 10260

aattgccctc cttttacccc taccatgagc cctacaaaca actaacctgc cactaatagt 10320

tatgtcatcc ctcttattaa tcatcatcct agccctaagt ctggcctatg agtgactaca 10380

aaaaggatta gactgaaccg aattggtata tagtttaaac aaaacgaatg atttcgactc 10440

attaaattat gataatcata tttaccaaat gcccctcatt tacataaata ttatactagc 10500

atttaccatc tcacttctag gaatactagt atatcgctca cacctcatat cctccctact 10560

atgcctagaa ggaataatac tatcgctgtt cattatagct actctcataa ccctcaacac 10620

ccactccctc ttagccaata ttgtgcctat tgccatacta gtctttgccg cctgcgaagc 10680

agcggtgggc ctagccctac tagtctcaat ctccaacaca tatggcctag actacgtaca 10740

taacctaaac ctactccaat gctaaaacta atcgtcccaa caattatatt actaccactg 10800

acatgacttt ccaaaaaaca cataatttga atcaacacaa ccacccacag cctaattatt 10860

agcatcatcc ctctactatt ttttaaccaa atcaacaaca acctatttag ctgttcccca 10920

accttttcct ccgaccccct aacaaccccc ctcctaatac taactacctg actcctaccc 10980

ctcacaatca tggcaagcca acgccactta tccagtgaac cactatcacg aaaaaaactc 11040

tacctctcta tactaatctc cctacaaatc tccttaatta taacattcac agccacagaa 11100

ctaatcatat tttatatctt cttcgaaacc acacttatcc ccaccttggc tatcatcacc 11160

cgatgaggca accagccaga acgcctgaac gcaggcacat acttcctatt ctacacccta 11220

gtaggctccc ttcccctact catcgcacta atttacactc acaacaccct aggctcacta 11280

aacattctac tactcactct cactgcccaa gaactatcaa actcctgagc caacaactta 11340

atatgactag cttacacaat agcttttata gtaaagatac ctctttacgg actccactta 11400

tgactcccta aagcccatgt cgaagccccc atcgctgggt caatagtact tgccgcagta 11460

ctcttaaaac taggcggcta tggtataata cgcctcacac tcattctcaa ccccctgaca 11520

aaacacatag cctacccctt ccttgtacta tccctatgag gcataattat aacaagctcc 11580

atctgcctac gacaaacaga cctaaaatcg ctcattgcat actcttcaat cagccacata 11640

gccctcgtag taacagccat tctcatccaa accccctgaa gcttcaccgg cgcagtcatt 11700

ctcataatcg cccacgggct tacatcctca ttactattct gcctagcaaa ctcaaactac 11760

gaacgcactc acagtcgcat cataatcctc tctcaaggac ttcaaactct actcccacta 11820

atagcttttt gatgacttct agcaagcctc gctaacctcg ccttaccccc cactattaac 11880

ctactgggag aactctctgt gctagtaacc acgttctcct gatcaaatat cactctccta 11940

cttacaggac tcaacatact agtcacagcc ctatactccc tctacatatt taccacaaca 12000

caatggggct cactcaccca ccacattaac aacataaaac cctcattcac acgagaaaac 12060

accctcatgt tcatacacct atcccccatt ctcctcctat ccctcaaccc cgacatcatt 12120

accgggtttt cctcttgtaa atatagttta accaaaacat cagattgtga atctgacaac 12180

agaggcttac gaccccttat ttaccgagaa agctcacaag aactgctaac tcatgccccc 12240

atgtctaaca acatggcttt ctcaactttt aaaggataac agctatccat tggtcttagg 12300

ccccaaaaat tttggtgcaa ctccaaataa aagtaataac catgcacact actataacca 12360

ccctaaccct gacttcccta attcccccca tccttaccac cctcgttaac cctaacaaaa 12420

aaaactcata cccccattat gtaaaatcca ttgtcgcatc cacctttatt atcagtctct 12480

tccccacaac aatattcatg tgcctagacc aagaagttat tatctcgaac tgacactgag 12540

ccacaaccca aacaacccag ctctccctaa gcttcaaact agactacttc tccataatat 12600

tcatccctgt agcattgttc gttacatggt ccatcataga attctcactg tgatatataa 12660

actcagaccc aaacattaat cagttcttca aatatctact catcttccta attaccatac 12720

taatcttagt taccgctaac aacctattcc aactgttcat cggctgagag ggcgtaggaa 12780

ttatatcctt cttgctcatc agttgatgat acgcccgagc agatgccaac acagcagcca 12840

ttcaagcaat cctatacaac cgtatcggcg atatcggttt catcctcgcc ttagcatgat 12900

ttatcctaca ctccaactca tgagacccac aacaaatagc ccttctaaac gctaatccaa 12960

gcctcacccc actactaggc ctcctcctag cagcagcagg caaatcagcc caattaggtc 13020

tccacccctg actcccctca gccatagaag gccccacccc agtctcagcc ctactccact 13080

caagcactat agttgtagca ggaatcttct tactcatccg cttccacccc ctagcagaaa 13140

atagcccact aatccaaact ctaacactat gcttaggcgc tatcaccact ctgttcgcag 13200

cagtctgcgc ccttacacaa aatgacatca aaaaaatcgt agccttctcc acttcaagtc 13260

aactaggact cataatagtt acaatcggca tcaaccaacc acacctagca ttcctgcaca 13320

tctgtaccca cgccttcttc aaagccatac tatttatgtg ctccgggtcc atcatccaca 13380

accttaacaa tgaacaagat attcgaaaaa taggaggact actcaaaacc atacctctca 13440

cttcaacctc cctcaccatt ggcagcctag cattagcagg aatacctttc ctcacaggtt 13500

tctactccaa agaccacatc atcgaaaccg caaacatatc atacacaaac gcctgagccc 13560

tatctattac tctcatcgct acctccctga caagcgccta tagcactcga ataattcttc 13620

tcaccctaac aggtcaacct cgcttcccca cccttactaa cattaacgaa aataacccca 13680

ccctactaaa ccccattaaa cgcctggcag ccggaagcct attcgcagga tttctcatta 13740

ctaacaacat ttcccccgca tcccccttcc aaacaacaat ccccctctac ctaaaactca 13800

cagccctcgc tgtcactttc ctaggacttc taacagccct agacctcaac tacctaacca 13860

acaaacttaa aataaaatcc ccactatgca cattttattt ctccaacata ctcggattct 13920

accctagcat cacacaccgc acaatcccct atctaggcct tcttacgagc caaaacctgc 13980

ccctactcct cctagaccta acctgactag aaaagctatt acctaaaaca atttcacagc 14040

accaaatctc cacctccatc atcacctcaa cccaaaaagg cataattaaa ctttacttcc 14100

tctctttctt cttcccactc atcctaaccc tactcctaat cacataacct attcccccga 14160

gcaatctcaa ttacaatata tacaccaaca aacaatgttc aaccagtaac tactactaat 14220

caacgcccat aatcatacaa agcccccgca ccaataggat cctcccgaat caaccctgac 14280

ccctctcctt cataaattat tcagcttcct acactattaa agtttaccac aaccaccacc 14340

ccatcatact ctttcaccca cagcaccaat cctacctcca tcgctaaccc cactaaaaca 14400

ctcaccaaga cctcaacccc tgacccccat gcctcaggat actcctcaat agccatcgct 14460

gtagtatatc caaagacaac catcattccc cctaaataaa ttaaaaaaac tattaaaccc 14520

atataacctc ccccaaaatt cagaataata acacacccga ccacaccgct aacaatcaat 14580

actaaacccc cataaatagg agaaggctta gaagaaaacc ccacaaaccc cattactaaa 14640

cccacactca acagaaacaa agcatacatc attattctcg cacggactac aaccacgacc 14700

aatgatatga aaaaccatcg ttgtatttca actacaagaa caccaatgac cccaatacgc 14760

aaaactaacc ccctaataaa attaattaac cactcattca tcgacctccc caccccatcc 14820

aacatctccg catgatgaaa cttcggctca ctccttggcg cctgcctgat cctccaaatc 14880

accacaggac tattcctagc catgcactac tcaccagacg cctcaaccgc cttttcatca 14940

atcgcccaca tcactcgaga cgtaaattat ggctgaatca tccgctacct tcacgccaat 15000

ggcgcctcaa tattctttat ctgcctcttc ctacacatcg ggcgaggcct atattacgga 15060

tcatttctct actcagaaac ctgaaacatc ggcattatcc tcctgcttgc aactatagca 15120

acagccttca taggctatgt cctcccgtga ggccaaatat cattctgagg ggccacagta 15180

attacaaact tactatccgc catcccatac attgggacag acctagttca atgaatctga 15240

ggaggctact cagtagacag tcccaccctc acacgattct ttacctttca cttcatcttg 15300

cccttcatta ttgcagccct agcaacactc cacctcctat tcttgcacga aacgggatca 15360

aacaaccccc taggaatcac ctcccattcc gataaaatca ccttccaccc ttactacaca 15420

atcaaagacg ccctcggctt acttctcttc cttctctcct taatgacatt aacactattc 15480

tcaccagacc tcctaggcga cccagacaat tataccctag ccaacccctt aaacacccct 15540

ccccacatca agcccgaatg atatttccta ttcgcctaca caattctccg atccgtccct 15600

aacaaactag gaggcgtcct tgccctatta ctatccatcc tcatcctagc aataatcccc 15660

atcctccata tatccaaaca acaaagcata atatttcgcc cactaagcca atcactttat 15720

tgactcctag ccgcagacct cctcattcta acctgaatcg gaggacaacc agtaagctac 15780

ccttttacca tcattggaca agtagcatcc gtactatact tcacaacaat cctaatccta 15840

ataccaacta tctccctaat tgaaaacaaa atactcaaat gggcctgtcc ttgtagtata 15900

aactaataca ccagtcttgt aaaccggaga tgaaaacctt tttccaagga caaatcagag 15960

aaaaagtctt taactccacc attagcaccc aaagctaaga ttctaattta aactattctc 16020

tgttctttca tggggaagca gatttgggta ccacccaagt attgactcac ccatcaacaa 16080

ccgctatgta tttcgtacat tactgccagc caccatgaat attgtacggt accataaata 16140

cttgaccacc tgtagtacat aaaaacccaa tccacatcaa aaccccctcc ccatgcttac 16200

aagcaagtac agcaatcaac cctcaactat cacacatcaa ctgcaactcc aaagccaccc 16260

ctcacccact aggataccaa caaacctacc cacccttaac agtacatagt acataaagcc 16320

atttaccgta catagcacat tacagtcaaa tcccttctcg tccccatgga tgacccccct 16380

cagatagggg tcccttgacc accatcctcc gtgaaatcaa tatcccgcac aagagtgcta 16440

ctctcctcgc tccgggccca taacacttgg gggtagctaa agtgaactgt atccgacatc 16500

tggttcctac ttcagggtca taaagcctaa atagcccaca cgttcccctt aaataagaca 16560

tcacgatg 16568

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